The long term goal of the present project is to identity the specific residues or sequences which contribute local and non-local interactions (LIs and NLIs) stabilizing the early intermediates of the folding pathway of a globular protein. The working hypothesis is that the gene sequence contains the coded program for the process of folding, (the "second genetic code"). Deciphering the protein folding code will help us better understand genetic defects, cancer and rational drug design. The assumption underlying this project is that the early intermediates which are programmed by specific sequence messages direct the folding path way and are responsible for the major reduction of chain entropy. The specific aims of the present phase of the project are to search for the earliest structural events of the folding pathway. A method based on time resolved dynamic energy transfer (ET) measurements has been developed. Intra- and inter-segmental end-to-end (EED) distributions and diffusion coefficients (Dis) are determined by global analysis of fluorescence decay curves of pairs of probes attached to selected sites on the protein backbone. By using site-directed mutagenesis and site-specific chemical labeling, protein derivatives will be prepared, each one of them designed to test a specific hypothesis. The high time and distances resolution (sub-nanoseconds and 10 to 80 Angstroms respectively) and sensitivity of the measurements, make it most suitable for detection of the backbone conformational distributions in the partially folded states, which cannot be determined by most other structure determination methods. Bovine pancreatic trypsin inhibitor (BPTI) is being used as the model protein. Double labeled BPTI derivatives designed for detection of LIs and NLIs will be prepared. The EED distributions between the labeled sites will be determined by laser based picosecond ET measurements in reduced BPTI under fully unfolding, partially unfolding and folding conditions. The order of formation of the earliest intra-and inter-segmental structures (secondary structures and folding initiations structures (FISs)) will be determined. The kinetics of the development of local structures in the reduced BPTI will be determined by the ET-stopped flow experiment. The role of NLIs and Lls in the compactization step will be analyzed. The question of whether formation of secondary structures occurs early will be investigated using comparative measurements with reference to statistical coil model peptides. This approach will enable detection of minor conformational biases by weak interactions between specific side chains, which might nevertheless be crucial in selection of the direction of folding. Double labeled model protein fragments will be prepared and measured in order to determine the contributions of specific Lls and NLIs, which initiate folding prior to the cooperative transition. By virtue of the high sensitivity and selectivity of the signals from the external labels, the same method will be further developed to detect folding intermediates under conditions closer to those found in vivo.